Recombinant DNA Technology Overview
10 Questions
0 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary purpose of adding alkaline phosphatase when working with a vector and restriction endonuclease?

  • To prevent the vector from re-annealing without the DNA insert. (correct)
  • To increase the efficiency of DNA ligase.
  • To generate compatible sticky ends on the vector.
  • To facilitate the breakdown of the phosphodiester bonds.

    • What limitation is associated with plasmid vectors in DNA cloning?

  • Challenges in generating sticky ends for ligation.
  • Restricted insert size and copy number problems. (correct)
  • Inability to transfect mammalian cells.
  • Limited expression of proteins due to high transcription rates.

    • What is a major advantage of using blunt end ligation with polylinkers?

  • It allows the use of multiple restriction enzymes flexibly. (correct)
  • It ensures higher insert sizes in plasmid vectors.
  • It uses enzymes with complete sticky compatibility.
  • It requires no ligase in the reaction.

    • Which type of ends generated by restriction enzymes require additional nucleotide addition to create compatibility?

    <p>Incompatible cohesive termini.</p> Signup and view all the answers

    Which enzyme is primarily responsible for sealing nicks in DNA after the insert has been added to the vector?

    <p>DNA ligase.</p> Signup and view all the answers

    What key discovery related to restriction endonucleases contributed to the understanding of DNA manipulation?

    <p>Some bacteria were found to be resistant to viral infection.</p> Signup and view all the answers

    Which type of restriction endonuclease is considered more favorable for genetic engineering?

    <p>Type II because it cuts at specific recognition sequences.</p> Signup and view all the answers

    What is the primary reason that restriction enzymes do not digest chromosomal DNA within bacterial cells?

    <p>The bacterial DNA is modified to prevent recognition by the enzymes.</p> Signup and view all the answers

    Which step in genetic engineering involves the insertion of the gene into a vector?

    <p>Cutting the DNA into fragments using restriction enzymes.</p> Signup and view all the answers

    How are restriction enzymes named based on their origin?

    <p>Using the genus and species of the bacteria they are derived from.</p> Signup and view all the answers

    Study Notes

    Recombinant DNA Technology

    • Biotechnology: This is a not new concept, Karl Ereky coined the term in the early 1900s.
    • Key Milestones:
      • 1953: Watson and Crick described the structure of DNA.
      • 1970s: Discovery of restriction endonuclease (EcoR1).
      • 1990: Human somatic cell gene therapy trials were approved in the US. Before this, genetically manipulating humans was illegal.
      • 2001: The human genome sequence was published.
      • CRISPR: NIH approved its safety in a clinical trial. It has proven effective in treating specific diseases but raises ethical concerns due to potential harm.

    Genetic Engineering Steps

    • Prepare the Gene:
      • Purification: Remove proteins and RNA from chromosomal DNA using enzymes (e.g., proteases, RNAse).
      • Phenol-Chloroform Extraction: Further purify DNA using this method.
      • Purity Check: Use spectrophotometry (260-280nm) to assess DNA purity.
    • Cutting DNA:
      • Restriction Enzymes: These enzymes cut DNA at specific recognition sequences.
      • Discovery: They were discovered because some bacteria were resistant to phage infection. These bacteria contained enzymes that fragmented the viral DNA, preventing phage replication.
    • Types of Restriction Enzymes:
      • Type I and III: Not preferred because they cut 1000 nucleotides from the restriction site and have combined methylase and nuclease activities. This makes their cuts unpredictable.
      • **Type II: ** Preferred for having separate methylase and nuclease activities, cutting directly at the recognition sequence. This allows for predictable cuts.
    • Naming Convention:
      • First Letter: Genus of the bacteria.
      • Second Two Letters: Species of the bacteria.
      • Strain: If applicable.
      • Order of Discovery:
      • Example: BamH1 (Bacillus amyoliquefaciens, strain H, first discovered)
    • Why Bacterial DNA Doesn't Get Cut:
      • Bacterial chromosomal DNA is methylated, preventing restriction enzymes from cutting it.
    • Properties of Type II Enzymes:
      • Short Recognition Sequences: 2,4,6, or 8 nucleotides long. They are palindromic, meaning they read the same backward and forward.
      • Types of Ends:
        • Sticky Ends (Overhangs): EcoR1 and BamH1 create these ends which are compatible and easily reanneal.
        • Blunt Ends: HaeIII creates these, which are not compatible with sticky ends and require more energy for reannealing.
        • Incompatible Cohesive Termini: HindIII and XbaI have overhangs that aren't completely compatible, requiring additional nucleotides for reannealing.
    • Sticky End Advantages:
      • The same enzyme can be used to cut the vector and insert DNA, making it easy to retrieve the insert from the plasmid.
      • DNA Ligase: This enzyme uses ATP to seal the nicks in the DNA after the insert is integrated.
      • Preventing Vector Re-annealing: Alkaline phosphatase can be added to the mixture to prevent the vector from re-annealing without the insert.
    • Blunt End Advantages and Disadvantages
      • Blunt End Ligation: Used to create Polylinkers (Multiple Cloning Sites - MCS) with various enzyme recognition sequences.
      • Disadvantages: Re-annealing is less efficient, requiring higher concentrations of DNA ligase.
    • Incompatible Cohesive Termini:
      • Disadvantages: These ends need filled-in nucleotides to make them compatible and re-anneal.
    • Insert Gene into Vector:
      • Vectors: DNA carriers used to deliver new genes into cells. They are used for cloning, protein expression, subcloning, and sequencing.
      • Types of Vectors:
        • **Plasmid Vectors (Circular): ** Maximum insert size 6-12kb.
        • **Bacteriophage Vectors (Linear): ** Maximum insert size 25kb.
      • Limitations:
        • Insert Size Restrictions: Vectors have a maximum size that they can accommodate.
        • Expression Limitations: Bacterial vectors might not have the necessary transcription factors for efficient protein expression.
        • Copy Number Problems: Some vectors have limitations on the number of copies they can produce.
        • Host Range: Most vectors are limited to bacteria or yeast.

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Description

    Explore the fascinating milestones and steps involved in recombinant DNA technology. This quiz covers historical advancements, key techniques in genetic engineering, and the ethical considerations surrounding innovations like CRISPR. Test your knowledge and understanding of biotechnology and genetic manipulation.

    More Like This

    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser